contents

1. Introduction

2. Summary of the Experiment

2.1. Realizing the Payoff Matrix of Prisoner A coli

2.2. Realizing the Payoff Matrix of Prisoner B coli

3. Results

3.1. Realizing the Payoff Matrix of Prisoner A coli

3.2. Realizing the Payoff Matrix of Prisoner B coli

4. Materials and Methods

4.1. Construction

4.2. Assay Protocol

4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

4.2.2. 3OC12HSL concentration-dependent CmR expression assay

4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag

4.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

5. Reference

1. Introduction

　　　　　　

We genetically engineered two prisoner coli, Prisoner A and Prisoner B. They are able to cooperate or to defect. The genetic circuits, with the improved chloramphenicol resistant protein(CmR) part, of Prisoner A and B are shown in Fig. 3-2-1-1. Our goal in this project is to realize the payoff matrix (Fig. 3-2-1-2) with the four types of growth inhibition. Using the improved plasmids we constructed, our E.coli version payoff matrix is realized through wet experiments.

Fig. 3-2-1-1. C4HSL-dependent CmR expression

　　　

2. Summary of the Experiment

2.1. Realizing the Payoff Matrix of Prisoner A coli

The cell growth of the Prisoner colis containing the pairs of plasmids (1) and (2) in the lower chloramphenicol (Cm) concentration (75 microg/mL) were measured and the payoff matrix was realized.

Fig. 3-2-2-1. Cells for the experiment to realize the payoff matrix of Prisoner A coli

2.2. Realizing the Payoff Matrix of Prisoner B coli

We have prepared a new pair of plasmids, (3) and (4), as in the following (Fig. 3-2-2-2.). The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and also in the lower 3OC12HSL concentration were measured and the payoff matrix was realized.

Fig. 3-2-2-2. Cells for the experiment to realize the payoff matrix of Prisoner B coli

　　　　　　

Using the pair of plasmids we constructed, our E.coli version payoff matrix with the four types of growth inhibition was replicated through wet experiments. The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4), grown under the lower Cm concentration and also with the lower 3OC12HSL concentration, were observed.

3. Results

3.1. Realizing the Payoff Matrix of Prisoner A coli

The cell growth of the Prisoner colis containing the pairs of plasmids (1) and (2) in the lower chloramphenicol (Cm) concentration (75 microg/mL) were measured after an eight hour incubation. The payoff matrix was realized as the following.

Fig. 3-2-1-1. C4HSL-dependent CmR expression

3.2. Realizing the Payoff Matrix of Prisoner B coli

　　　　　　

The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and in the lower 3OC12HSL concentration were measured. The payoff matrix was realized as the following.

Fig. 3-2-1-1. C4HSL-dependent CmR expression

4. Materials and Methods

4.1. Construction

-Strain

　　　　　　

All the samples were JM2.300 strain.

-Plasmids

　　　　　　

(1) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-4-1.

　　　　　　

(2) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) +promoter less_lasI (pSB3K3)

Fig. 3-1-4-2.

　　　　　　

(3) Pcon_rhlR_TT_Plux_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-4-3.

　　　　　　

(4) Pcon_rhlR_TT_Plux_CmR (pSB6A1) +promoter less_lasI (pSB3K3)

Fig. 3-1-4-4.

　　　　　　

(5) Negative control1: Pcon_rhlR_TT_promoter less_CmR (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-1-4-5.

　　　　　　

(6) Negative cotrol2:Pcon_rhlR_TT_promoter less_CmR (pSB6A1) +promoter less_lasI (pSB3K3)

Fig. 3-1-4-6.

4.2. Assay Protocol

4.2.1. C4HSL-dependent CmR expression assay

-samples
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋ promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1) ＋ promoter less_lasI (pSB3K3)#2
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_promoter less_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
   ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)

4.2.2. C4HSL-dependent CmR expression assay (With an ssrA tag)

-samples
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2
Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_promoter less _CmR (pSB6A1)＋promoter less_lasI (pSB3K3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
   ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (3 microL of 100 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure optical density every hour. (If the optical density is over 1.0, dilute the cell medium to 1/5.)

4.2.3. Chloramphenicol-dependent Growth Assay with ssrA tag

-samples
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1 mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (6 microL of 25 microg/mL) + 99.5% ethanol (6 microL)
   ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (9 microL of 25 microg/mL) + 99.5% ethanol (3 microL)
   ③) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (12 microL of 25 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure the optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

4.2.4. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

-Samples
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋Plac_lasI (pSB3K3)#2
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#1
Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1)＋promoter less_lasI (pSB3K3)#2

-Procedure
1. Prepare overnight cultures for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp (50 microg/mL) and Kan (30 microg/mL) and grow the cells at 37°C until the observed OD590 reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5. Add 30 microL of suspension in the following medium.
   ①) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (75 microg/mL)
   ②) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (75 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
7. Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

5. Reference

　　　　　　

1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619